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Ilan Riess, Mark Gutierrez, Joe Reynolds, Deepak Lamba; Direct reprogramming of Human Fibroblasts into RPE and Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1171.
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© ARVO (1962-2015); The Authors (2016-present)
The goal is to generate directly RPE and photoroeceptors in vitro from human fibroblasts cell lines without the need to bring the cells into a stem cell phase, in order to create cell source replacement for patients with Age Related Macular Degeneration and Retinitis Pigmentosa.
Cell Culture: Human fibroblasts were maintained in DMEM-F12 (50:50) with 20% FBS, 0.2% BME, 1% MEM-NEAA, pen-strep and 3ng/ml of FGF. Viral constructs and infections: virus was prepared as follow: 293T cells were transfected with a mixture of viral plasmid (FUW-Ascl1, Otx2, Blimp, Mitf3, Pax6, Otx2, rTTa) and packaging constructs expressing the viral packaging functions and the VSV-G protein. Medium was replaced 24hrs after transfection and viral supernatants were collected at 48hrs and 72hrs. After filtration, supernatants were pooled and fibroblasts were incubated with viral supernatants and fresh media at a ratio of 1:1 for 24 hours. Cells were subsequently cultured in cell-specific differentiation medium containing doxycycline (2 μg/mL) to activate expression of the transduced genes. Immunocytochemistry: Cells were fixed with 2% paraformaldehyde for 10 minutes. Washed with PBS and then permeabilized and saturated with block solution (10μl Triton-X, 1% FBS in 9ml 1X PBS) for 1 hour. Primary antibodies were left overnight (Ascl1, Blimpl, Otx2, Pax6, Mitf3, Oct4). The next day secondary fluorescent antibodies were used and cells were stained for DAPI. Real Time Quantitative PCR Analysis: RNA was extracted from the cultures using TriZol (Invitrogen). cDNA was generated using iScript cDNA Synthesis Kit (BIO-RAD). RT PCR was performed using the following protocol: 1) 95.0°C for 2’, 2) 95.0°C for 5”, 60.0°C for 10”, 72.0°C for 20”, for 39 cycles, 3) Melt Curve 65.0 to 95.0°C, increment .5°C for 5” (Figure 4). Normalization was done with β-Actin.
First we validated the expression of transduced genes by immunocytochemistry and RT-PCR. Upon further culture (4-6 weeks) we assessed transdifferentiation into an immature state of RPE or photoreceptors. We found expression of cell-type specific markers by immunostaining. We further confirmed by RT-PCR, the transdifferentiaion fate by the mRNA expression in each specific cell type.
Transdifferentiation of human fibroblasts into either RPE or photoreceptors can be achieved by activating specific primordial lineage genes and further induction by using specific medium.
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